Process for stimulating the growth of epidermal cells

ABSTRACT

A process for stimulating the growth of human epidermal cells, applications of said process and products applying the said process. 
     A process for stimulating the growth of epidermis cells. 
     The epidermis cells are contacted with an aqueous saline extract (RE) of ocular tissues. The process is especially applied for human epidermis cells. 
     Application to the study of chromosomes of the human epidermis cell, particularly for the detection of natural or induced chromosome anomalies, and to the production of grafts, and especially autografts, usable on man, for example in the treatment of burns, cosmetic and diagnostic products.

This invention relates to processes and products applicable to the humanskin and more particularly to the adult human epidermis. Its object isnotably a process for stimulating the growth of human epidermal cells,particularly those of adults, as well as cosmetic, pharmaceutical anddiagnostic products using the said process.

The skin is the principal tissue exposed to the mutagenic effects ofchemical and physical environmental agents. This is particularly true ofradiations of every sort. The skin is composed of tissues of mesenchymalorigin (the derma and the blood vessels) and tissues of ectodermicorigin (the epidermis). Now, although it is known to extract mesenchymalcells (fibroblasts) from the dermis to culture them and to study themutagenic and carcinogenic effects of chemical and physicalenvironmental factors, it is difficult to extract them from theepidermis. One of these difficulties had not yet been overcome. This isthe application of processes of cytogenetic examination to culturedadult epidermal cells, which involves on the one hand the preparationand analysis of chromosomes, and on the other, the notation of sisterchromatid exchanges (SCE). In point of fact, although a few rarepublications mention succinct chromosomic investigations, there is noexhaustive chromosomic investigation, particularly in so far asheriditary human skin diseases are concerned and, to the best of theapplicant's knowledge, no bibliographical references exist with respectto SCE.

A series of recent investigations suggests that the increase of SCEs isa sensitive and accurate indication of the mutagenic effect of achemical product or of a radiation. The application of SCE-revealingtechniques to adult human epidermal cells is, therefore, unquestionablyimportant.

The object of the invention is, notably, a process applicable to thepreparation and the analysis of chromosomes and to the preparation andthe notation of SCEs. According to the invention, it is possible toinduce mitotic multiplication of these cells on a normally hostile glasssubstrate.

The invention also makes it possible to provide a solution to all theproblems connected with the growth and culture of epidermal cells,particularly in man and especially in adults. Thus, apart from theabovementioned cytogenetic application, the invention can also be usedin the treatment of burns, in the fields of cosmetics and of cutaneouspharmacology and for long-term culture of epidermal cells.

The present invention calls upon previous work reported by ARRUTI C. andCOURTOIS Y. Exptl. Cell. Res. 117, 283-292 (1978). These authorsestablished that a retinal extract, referred to by the abbreviation RE,was a growth-promoting factor for cultured epithelial lens cells. Theskilled artisan can, if needs be, refer to this article to obtain thenecessary information on this retinal extract RE, and the propertieswhich have been observed. The authors used a total retinal extractobtained by extraction with an aqueous salt solution, with a pH 7.2phosphate buffer such as the type sold by FLOW Laboratories Ltd. Thebovine retinas are placed in intimate contact with such a solution.After a certain number of physical centrifugation and filtrationtechniques the retinal extract RE is isolated. However, the observationsreported in this article are limited to the growth of epithelial lenscells.

Certain documents relating to the prior technique describe work onextracts obtained from animal ocular tissue.

French Pat. No. 71.13.756 (publication 2.134.088) describes a processfor obtaining a total bovine eyeball extract. The series of stepsinvolves the action of merthiolate, of an alcaloid and of "Celite".

The alcoholic extract so obtained is applied in the field ofophthalmology. It is not, therefore, an aqueous saline solution, and itsuse for the growth of epidermal cells is not mentioned.

The article by B. S. Kasavina et al. (Columbus, Ohio, USA) and SU Pat.No. 130.159 of the July 15, 1960 cited in Chemical Abstracts Vol. 55,No. 5 (Mar. 6, 1961) No. 4892c describe the preparation of a productcontaining hyaluronic acid by extraction from the vitreous humour ofcattle eyes, and its application to the treatment of infected wounds.The product is a chloroformic extract.

The article by M. A. Karasek in J. Invest. Dermatol. 1972, 59 (1),99-101, cited in Chemical Abstracts Vol. 77, No. 11 (Sept. 11, 1972) No.73 097 m, p. 305, deals with the in vitro growth of epidermal cells inthe presence of a certain number of factors. It is established thathyaluronic acid did not stimulate epithelial cells. This article alsoshows that it is impossible to forsee the effect of a determined factoron epidermal cell growth.

It is therefore clear that the prior technique does not teach a factorconsisting of an aqueous extract of ocular tissue that is effective instimulating the growth of epidermal cells, particularly adult,especially human, epidermal cells.

According to the present invention, the growth of cultured epidermalcells can be stimulated irrespective of the age and the species of thedonor, by using the growth factor RE. Also according to the invention,the growth factor RE is effective on adult, and particularly on humanepidermal cells.

In its most general form, the invention relates to a process forstimulating the growth of epidermal cells particularly adult cells, withan aqueous salt extract of ocular tissue, i.e. eyeball tissue, (or REextract).

To obtain the active RE agent, the technique described in theabovementioned article by C. Arruti and Y. Courtois can be used. Thestarting material, namely bovine retinas, is easily available andabundant.

The expression "ocular tissue extract" designates a product that can beextracted from various tissues of the eye, such as the choroid, the irisand the vitreous humour. This definition does not include thecrystalline lends and the aqueous humour, tissues that do not containRE.

To effect extraction, any aqueous salt solution is used, buffered to apH of approximately 7.2 and capable of providing an aqueous extractcontaining the RE.

Surprisingly, it was ascertained that by using the RE factor, accordingto the invention, in adult human epidermal cell cultures, it becamepossible to provide a solution to problems that had not been solved bythe prior technique. For example, the invention makes it possible toeffect chromosome analysis and the evaluation of SCEs in in vitrocultures of adult human keratinocytes, through the activation of themitotic activity of keratinocytes resulting from the use of RE.

The activator effect of RE on the growth of epidermal cells will beillustrated below. An increase in the surface covered in a given numberof days by the same number of seeded cells is observed. The cell densityper unit area revealed by staining is greater for the treated cells.This stronger staining is not due to an increase in the height of thecells but to an increase of mitotic activity.

As has already been stated hereinabove, it was impossible to preparemetaphasic chromosomes of adult human cells in the absence of cellulargrowth activities; see, for example, Green H. Cell. 15, 801-811 (1978).Cells on glass slides in an ordinary culture medium (TCM) in thepresence of RE makes it possible to obtain a number of mitoses necessaryfor the requirements of the invention. According to the factsestablished in the prior art, the primary keratinocyte cells tend topile up and to keratinize. Thus, somme metaphases are situated under oneor more layers of cells, which prevents chromosome dispersion. Part ofthe "spontaneous" mitoses is therefore lost and too few remain. On theother hand, the addition of RE induces a marked increase in mitoticactivity which, in combination with a 15 hour treatment with colchicinesalt, available under the trade name of "Colcemid" (Gibco), makes itpossible to prepare a sufficient number of well spread out metaphases.An average of 20 analysable metaphases per slide can be obtained inroutine operation.

The effect of RE on adult epidermal cells differs from the effect of theproduct EGF (Epidermal Growth Factor), see COHEN S. and ELLIOTT G. A. J.I, vest. Dermatol., 40, 1-5 (1963), and COHEN S. in Hormones andDevelopment, Hamburgh M. and Burrington E. J, ed. Meredith Corp. N.Y.,753-766 (1971), which is essentially active on foetal cells or cellsfrom newborn infants. Furthermore, these two mitogenic agents differcompletely by their action on other target cells.

It has also been established that RE was not mutagenic, which is a veryimportant characteristic for adult epidermal cells. This property can,moreover, be used for the detection of the action of mutagens.Experiments have shown that the increased growth of the cell culture,was indeed due to an actual multiplication of keratinocytes, and not tothat of possible fibroblasts which might have been detached from thedermis during trypsinization. For example, phase contrast microscopeexamination reveals that the cultures are composed of a single celltype, having the appearance of an epithelium. Furthermore, the reactionto the leucine-aminopeptidase (LAP)enzyme marker of cells of mesenchymalorigin in vivo (NACHLAS N. M., CRAWFORD D. T. and SELIGMAN A. M. J.Histochem. Cytochem. 1957, 5, p. 264-278) and in vitro (FRITSCH P., DIEME. Arch. Derm. Forsch 243, 364-372 (1972), REGNIER M., DELESCLUSE C. andPRUNIERAS M., Acta Dermatovener, 53, 241-247 (1973)) was used to detectdermic fibroblasts, and was completely negative.

The RE growth potentialization effect on adult human epidermal cells maybe put to many applications.

In one application, given as an example, the invention makes it possibleto effect chromosomic analysis and assessment of SCEs in in vitrocultures of adult human keratinocytes. Well dispersed metaphasicchromosomes can be prepared in a number sufficient for karyotypicanalysis and also SCEs can be counted in this cell type.

Cytogenic methods, and particularly the techiques for assessing sisterchromatid exchanges (SCE), have recently been developed (see PERRY P.and Wolff S. Nature 251, 156-158 (1974)). This last technique is ofspecial interest in cutaneous pharmacology as the number of exchanges isdirectly connected with mutagenesis (PERRY P. and EVANS H. J., Nature258 121-125 (1975) and CARRANO A. V., THOMPSOM L. H., LINDL P. A. andMINKLER J. L., Nature 271, 551-553 (1978). For this reason SCE countingis invaluable for estimating the carcinogenic potential of a cosmeticproduct, or a therapy or, more generally, of a voluntary or accidentalexposure to chemical or physical enviromental factors (all forms ofradiations). In the literature, there is very little information on thecytogenetics of epidermal cells and there is no work reported on SCEs.This dearth of information is doubtless due to three major difficulties.

The first lies in the spreading of chromosomes. Trypsin is usually usedto harvest cells in mitosis and to disperse chromosomes. However, toharvest adult cultured keratinocytes, EDTA must be used in addition totrypsin (RHEINWALD, J. G. and GREEN H., Cell., 6, 331-344 (1975)) andEDTA interfers with cell swelling in hypotonic solution, which is afundamental requisite for good chromosome spreading. The chromosomes canbe dispersed satisfactorily by directly treating the culture in situ,but the experimentor then comes up against a second difficulty, becauseglass is the best surface for chromosome spreading, but it is a badsubstrate for attaching keratinocytes (LIU S. C. and KARASEK M. J.Invest. Dermatol. 71, 157-162 (1978)).

The third difficulty is that, to obtain SCE, two cycles of consecutivereplication of the same cells are necessary to visualize thedifferential labelling of chromatids with the technique of substitutionwith bromo deoxyuridine (BUdR) (WOLFF S. and PERRY P. Chromosome 48,341-353 (1974)--KATO H. nature 251, 70-72 (1974)). The culture musttherefore possess a high level of mitotic activity, which is not thecase of adult human keratinocyte cultures on a glass substrate.

For the preparation of SCEs it is most important that an appreciablenumber of cells should divide synchronically. As was said hereinabove,two cell cycles are necessary for a same cell to have one of its DNAstrands bisubstituted with BUdR, an indispensable preliminary tovisualization of SCEs. Furthermore, both these cycles must for practicalreasons, take place in a reasonable period of time (50-70 hours).

The epidermal cells in primary culture are not synchronized and thecells that replicate at a given moment do not necessarily belong to thesame group of replicating cells. This means that only a portion of thereplicating cells might undergo a second replication cycle during therelatively short period of time available. The great advantage of RE isthat it induces sufficient synchronization to enable about 50% of themetaphases to be bisubstituted in about 60 hours. The SCEs can then becounted on about 10 metaphases per slide.

According to one embodiment, a preliminary normal culture of epidermalcells is effected without the growth factor. In a period of between 5and 10 days, for example at the end of 8 days, a first effective amountof RE is introduced. The following day, or 48 hours later, a furtherapplication of RE is made to obtain a second cell replication.

However, this embodiment is only given by way of example. As a variant,the RE can be introduced at the beginning of the operation, but caremust be taken to see that cell development is synchronized. If thisvariant of the procedure is adopted, the RE must be introduced into thecell culture at the start and the culture then arrested to enablesynchronization of the cells.

The invention therefore relates to diagnostic products, making itpossible to study adult human epidermis cell chromosomes. Such productsand the processes for embodying them can make it possible to detectnatural or induced chromosome anomalies. Inductive factors are, notably,irradiations as, for example, those resulting from the action of UVrays, or from accidential phenomena, as well as all radiations, such asgamma rays, X rays, neutrons, etc. Among the disorders that can bedetected the following may be mentioned: genetic disorders, psoriasisand all types of skin cancers. The adult keratinocyte cells arerecognized as target cells for skin carcinogenesis. The advantage of theinvention is that it permits direct study of human epidermal cellbehaviour, and that it does not require in vivo experimentation, as wasthe case in the known technique, on partial cell samples, such asfibroblasts.

The invention can be applied, as a preventive measure to study thebehavior of skins in certain environments, for example to choose thepersonnel to work in a nuclear environment.

In such applications, the invention makes use of the fact that RE doesnot induce mutagenesis. For example, when chromatid exchanges arecounted, the exchange rate is observed to be lower that 3 per mitosis,which corresponds to a very low figure that is usually called"spontaneous chromatid exchanges".

It has also been established that the RE used according to the inventionis not toxic. When the dose of RE is doubled the cells do not die. Thisproperty is obviously very important for the applications which will nowbe described.

From another point of view, the invention is applicable to a process formaking grafts. It is known that, for the treatment of burns, it isimportant to be able to use the remaining intact skin in order to avoidthe problems arising from the immune defenses of the receiver in thecase of heterologousgrafts. It is therefore preferable to take pieces ofskin from the burn victims themselves to effect autografts. The tissueremoved is then separated mechanically in order to multiply by two orthree the area covered as the result of the growth of the excised skin.An important improvement has been made to this technique (see IGEL H. J., FREEMAN A. E., BOECKMANN C. R. and KLEIFLED K. L. Arch. Surgery 1974,108, 724-729).

According to the technique proposed by these authors, pieces of skin areexcised in the form of small fragments for subsequent autografting. Thefragments are split 3 to 5 weeks prior to grafting, and it is thereforeadvisable to cultivate this skin. The major problem lies in thetransport of epidermal cells. The skin fragments are placed on flags ofslaughtered pig skin. Some three weeks later the pig skin is coveredwith an epidermis culture. This is an advantageous technique as itenables pieces of skin to be taken from the patient's own body.Furthermore, there are no undesirable reactions to the pig skin. An areaof 20 to 40 times the area of the original fragments is thus obtained.

There are, however, certain drawbacks to this technique. The portion ofthe graft placed on the patient's tissue is in contact with the latterthrough the dead epidermis of the pig skin carrying the culturedepidermis. The presence of the pig skin constitutes an undesirableforeign body. Another drawback lies in the mechanical splitting of theskin used as the graft, as the fragments are necessarily fairly large.It is desirable to obtain ultimate splitting, which, in an ideal case,consists of a single cell. An even more advantageous technique has beenproposed recently, derived from the above-mentioned IGEL and FREEMANtechnique, and in which the graft is split by an enzymatic process 1 to2 weeks before being grafted. This period is used to effect in vitroproliferation of the epidermal cells. In order to provide these culturedepidermal cells with a substrate permitting them to proliferate invitro, and then to be transplanted, the epidermis was removed from thinflaps of human or pig skin. The flaps of derm so obtained were killedand conserved by freezing. The advantage of flaps of dermis with theepidermis removed is that they carry their "lamina densa", in otherwords a layer of collagen on which the epidermal cells fasten andproliferate. Recombined grafts are thus obtained formed of allo orxenogenic derm covered with autologous epidermis. Grafts of this sortmake it possible to multiply the area epidermized by about 80 in twoweeks. A technique of this sort was described, for example, in a reportmade at the 4th national meeting on care for burn victims. May, 21-22,1979 at La Baule, France.

Whatever the grafting technique used, it is essential to increase thespeed of proliferation of the epidermal cells. The advantage ofobtaining extensive cultures and, even more important, of being able toeffect the culture in a relatively short time, in, for example, a weekor less, in order to treat the patient effectively, is indeed obvious.

The invention can advantageously be applied to such a cell culture, asthe area covered by the growth of epidermal cells will be proportionalto the activity of the growth factor RE. The use of RE providesadvantages because, as was mentioned hereinabove, it does not inducemutagenic reactions. It is also established that epidermal cells aremodified after their growth in the presence of RE. The process of theinvention is, therefore, advantageously applied in the production ofgrafts for use in the treatment of burns.

By removing a small piece of skin, for example approximately 2 to 3 cm²,a suspension of epidermal cells can be obtained. As was previouslystated, culture can be initiated and the cell colonies grow sufficientlyin about 8 days. The addition of retinian extract RE for 2 consecutivedays makes it possible to obtain mitosis synchronization.

From still another point of view, the invention has to do with cosmeticapplications. Conventional cosmetic agents have an effect on the part ofthe body to be treated, for example, the skin or the hair. However, itis established that cosmetic formulae have an increasing tendancy tocontain agents that effect metabolism. Insofar as concerns applicationto the skin, for example, products that increase epidermal cell growthare sought, as it has been established that the said growth improves thesatiny appearance of the skin. Similarily, manufacturers are engaged inpreparing products that encourage hair growth when they are applied tothe hair or the scalp.

According to the invention, RE can be introduced directly into apresentation suited to cosmetic applications. The presentation must, ofcourse, be adapted to the cosmetic application sought, whether it be,for example, skin regeneration, the fight against signs of ageing or anapplication to the hair. Furthermore, RE can be used in more specificapplications, particularly for the treatment of deficient epidermalcells.

Most of the active agents known in this field cannot be used for thedirect treatment of the skin. Many of them are obtained from euphorbiaand are considered to be cocarcinogenic. Generalized application shouldtherefore be avoided. The epidermal growth factor E.G.F. appears to beinactive in vivo, or at least, no in vivo activity has beendemonstrated. In addition, it is expensive to produce. Thereforecosmetic applications of such a product on a large scale are not likely.The property of chlolera toxin to activate the skin vigorously is known,but such a product is difficult to manipulate.

According to the invention, on the contrary, RE can be used for itsactivity on the cells of the epidermis, taking advantage of the factthat it is not toxic.

Thus, the invention relates to a cosmetic composition characterized inthat it comprises, as active agent, RE. Such a composition can beapplied locally to the part of the body to be treated.

For the requirements of the invention, any of the vehicles alreadyproposed for application to the skin or the hair can be used, notablycompositions in the form of lotions, gels, creams, ointments, etc.Examples of compositions suited to local application are described by B.KAMMERAU et al. in Arch. Derm. Res. 255 31-42 (1976).

The amount of RE to be used in the compositions of the invention dependson the application considered. Of course, local application can berepeated to provide the necessary amount of active agent. Generallyspeaking rates of 50 to 400 μg of total proteins of RE per ml ofepidermal cell culture have been found to be satisfactory. Rates in therange of 50 to 100 μg are preferred.

The invention is also applicable to a long term epidermal cell cultureprocess.

Studies on adult human skin cells meet with many difficulties,especially when adult human epidermis cells are concerned. This is thereason why most experiments are conducted on non-epidermal cells,especially fibroblasts. Fibroblasts are known to possess growthproperties in culture that enable practical experiments to be conducted.However, growth of fibroblasts in culture is limited, especially in thecase of human cells. A sort of programming exists for human fibroblaststhat prevents them from doubling indefinitely. Then again, it is knownthat the number of fibroblast doublings is greater with embryonic cellsthen with adult cells. In the case of adults, the culture dies aftersome thirty doublings. Some animal cells, for example, mouse cells,appear to escape this rule, but, in the case of man, the only means ofaltering this natural growth limitation control is to introduce virusesor other exogenic substances into the cell culture.

The growth limitation already observed in connection with fibroblasts isstill more marked with epidermal cells. Cultures of newborn epidermiscells can be doubled 4 or 5 times, whereas cells from adult skin undergopractically no doubling.

The best system known at present for prolonging long-term cultures ofhuman epiderm was proposed in RHEINWALD and GREEN'S article mentionedabove. It consists in coculturing human epidermal cells with irradiated3 T 3 mouse fibroblasts as the nutritions substrate. These irradiatedcells have the property of enhancing the binding of human keratinocytesduring culture doubling, and of providing them with nutritionssubstances of conjunctive origin that increase their growth. Theirradiation blocks their own multiplication.

Moreover the 3 T 3 mouse cells have a somewhat inhibiting effect on thegrowth of human fibroblasts, thus enabling the authors to control thepurity of their cultures while starting with what may sometimes be veryheterogenous suspensions. During the various culture doubling steps anypossible human fibroblasts are removed by the action of the EDTA, at thesame time, as the 3T3 mouse cells; the human keratinocytes remainingattached to the substrate, subsequently to be detached by mechanical andenzymatic means. The long-term culture system can have added to itvarious growth factors, that is to say, products capable of stimulatingcell growth, and particularly that of cells extracted from adulttissues. In this connection, COHEN S., and ELLIOTT G. A. in J. Invest.Dermatol. 40 1-5 (1963) described a product capable of enhancing thegrowth of mouse epidermis cells. Culture experiments were conducted andthe authors (RHEINWALD J. C. and GREEN H. Nature 1977,265,421-424) foundthat this growth factor is effective on newborn epidermal cell cultures.

The question of the activating effect of EGF on the growth of adultepidermal cells remains to be solved. In culture, and in the presence of3T3 mouse cells, a certain activity on human cells, was reported. Usedalone on guinea pig cells, it proved to be ineffective. Finally, on thewhole subject, proof of activation is lacking.

Other activators have been described and, as a bibliographicalreference, mention may be made of the article by D. GOSPODAROWICZ inNature 249 (1974) 123. The author describes a growth factor designatedby the abbreviation F.G.F, but no indication can be found of thisproduct activity in the growth of human epidermal cells.

Choleraic toxin has also been studied in human keratinocyte cultures(GREEN H. Cell 1978, 15 801-811). Contrary to its action on many otherof the cells studied, this product strongly activates the growth ofhuman keratinocytes, particularly those of newborn humans. However, itis bound firmly to cell membranes, and this bond is non-reversible.

According to the invention, cells can be directly cultured on asubstrate, such as a plastic substrate, without the addition of othercells. The RE acts as the nutritive factor, its rather negative actionon fibroblasts making it possible for a culture, that has previouslybeen purified, that the amount of possibly contaminating fibroblasts bestabilized at a very small number. The life span of the culture in thusincreased in a very simple way. Furthermore, the unquestionable effectof RE on adult cells makes it a valuable mitogenic agent for thematerial which only reacts weakly to the previously described culturesystem with 3 T 3 mouse cells. The cytogenetic studies described abovefurther show that this product has no effect either on chromosomes or onthe exchanges of keratinocyte chromatids. It is not, therefore, apriori, suspected of having toxic or carcinogenic effects on thesecells.

By the use of RE according to the invention, adult human keratinocytecultures can thus be prolonged. Such a culture process is very easy toimplement. It further respects the normal nature of adult humankeratinocytes, only providing them with a mitogen nutritive supply ofphysiological animal origin.

The invention will now be illustrated in greater detail, while in no waybeing limited thereto, by experiments on adult human epidermal cellculture, and by definite examples relating to caryotypes and exchangesof sister chromatids on cultured adult human keratinocytes.

ORIGIN AND PREPARATION OF THE HUMAN SKIN

Adult human skin is removed during surgical operations.

The thin skin, removed with an electric dermatome, is placed directlyinto a bottle containing a solution of antibiotics (solution II, seebelow) and kept at +4° C. for up to 5 days.

The total skin, not cleansed of its fat, is placed in a jar containingsolution II. It is kept like this for 24 hours. The fat is then removedfrom the skin with a surgical knife, and firmly secured, derm downwards,on a cork mat washed with 70° alcohol. The skin is then cut finely bymeans of an electrokeratome adjusted to a thickness of 0.4 mm. Bearingin mind the variations in the thickness of the epidermis depending onthe donor's age and the site from which the skin was removed, thisthickness of 0.4 mm corresponds to the total epidermis plus a portion ofthe papillary layer of the dermis. This thinned-down skin is replaced insolution II and can be kept at +4° C. for up to 3 days.

The thin skin and the total skin so prepared are treated in an identicalmanner.

The antibiotic rinsing solutions are prepared in 500 ml of Eagle basemedium as follows:

SOLUTION I: antibiotic antimycotic product commercially available underthe name ABAM (GIBCO):

20 ml

Gentalline: 160 mg

SOLUTION II:

ABAM: 10 ml

Gentalline: 80 mg

SOLUTION III:

ABAM: 5 ml

All the cultures were carried out in the following medium: Dulbecco'sModification of Minimum Eagle Medium (Flow) in Hepes buffer, with theaddition of 20% foetal calf serum. 1% of ABAM (Gibco) was added to thetotal medium.

PREPARATION OF KERATINOCYTE SUSPENSIONS FROM TOTAL THINNED SKIN

The skin is passed successively through the three antibiotic solutions.

SOLUTION I: 30 minutes

SOLUTION II: 30 minutes

SOLUTION III: 3 times 10 minutes.

Between each bath it is blotted on a sterile gauze pad. The skin is thenstored in solution III.

For cutting up, the skin is spread, derm down, on the cap of a sterileglass Petri dish. It is cut into fragments measuring some 0.25 cm with asterile surgical knife.

The fragments are then placed in a 0.25% trypsin solution (Flow) in aDulbecco phosphate buffer free of calcium and magnesium (PBS-Flow), inamounts of about twenty fragments for 5 ml of trypsin solution in 50 mlconical tubes (Falcon 2070). The tubes are put in a refrigerator at +4°C. for one night. The next morning the fragments are picked out with abig sterile forceps and placed on the cap of a sterile glass Petri dish.They are spread out with the epidermis against the glass. The derm andepiderm are easily separated with two fine forceps. All the fragmentsare separated in this way and placed in a tube containing 5 ml PBS. Thecap is rinsed with 2 ml PBS which is added to the tube. Once the tubesare ready, they are vigorously agitated at 80 r.p.m. (Vortex), whichmakes it possible to detach the cells from the surface of the derm andto dissociate the lower layers of the epidermis. The PBS containing thederm+epiderm+dissociated cells is poured into a beaker through twolayers of sterile gauze, making it possible to retain the derm, theepiderm and the big cellular aggregates.

The suspensions so recovered are returned to the tubes and centrifugedat 800 r.p.m. for 10 minutes. The PBS is thrown away and the cellularbottoms are put into suspension again in 4 ml of complete medium pertube.

Cells are counted after dilution to 1/2 of an aliquot in Trypan Bluewith a Burker hematimeter.

STUDY OF THE STIMULATION OF ADULT HUMAN KERATINOCYTES BY RE (a) Trialswith 100,000 cells per cm².

Cell suspensions are adjusted in a complete medium to 400,000 cells perml and applied to two Costar type slides with 24 holes in amounts of 0.5ml of the suspension per hole.

The plates are placed in a drying oven at 37° C. for 24 hours (day 1);the medium is renewed every second day and, on one of the plates, 10 μlof RE, corresponding to 100 μg of protein, are added to all the holesevery day. The other plate is used as a control. On the 8th day theculture medium is thrown away from both plates, the cells are rinsed inPBS and fixed by Carnoy fixative (methyl alcohol: 3 volumes, aceticacid: 1 volume) for 20 minutes at ambient temperature, then dried in thedrying oven at 37° C. for one hour. The cultures are then stained(Giemsa RAL: 4 volumes; Sorenson phosphate buffer pH 6.7:4 volumes;deionized water: 92 volumes) for 10 minutes, rinsed with deionized waterand left on filter paper until completely dry.

(b) Trials using a variable number of cells

The cell suspensions are adjusted in a complete medium to 10⁵, 2.10⁵,4.10⁵, 8.10⁵, 12.10⁵, 16.10⁵ cells per ml. Each dilution is dividedbetween four holes in two plates in ascending order, at a rate of 0.5 mlper hole.

Two plates are thus filled and put into an oven at 37° C. The plates arethen treated exactly as in the preceeding trials (a), one of them beingsubjected to the action of RE, the other acting as control. Fixing andstaining are carried out on the 8th day in exactly the same way.

The stained surface visible in the holes and the intensity of stainingindicate the intensity of cell growth. In order to quantify this growthand to check that it is not a question of cells being better spread out,mitoses were counted.

In each hole of each plate, the number of mitoses present in fivemicroscropic fields at a magnification of ×250 were counted. The numberof mitoses per field were counted for each starting cell dilution, andthe results between the RE-treated cultures and the control ones werecompared.

The purity of the cultures was tested by submitting them to reactionwith leucine aminopeptidase (LAP) (See article by NACHLAS et al., op.cit.). This reaction was found to be negative, while it was positive fornonepidermal cells.

PREPARATION OF CHROMOSOME SPREADS

Cultures for cytogenetic trials were placed on glass slides in Leightontubes.

The Keratinocytes were adjusted to a concentration of 400,000 cells perml in a complete medium and divided up in amounts of 2 ml per tube inLeighton tubes with a glass slide on the flattened portion. The tubeswere then sealed and placed horizontally in an oven at 37° C.

The medium was changed after 48 hours and after 5 days culturing.

On the 8th day of incubation, 40 μl (400 μg of protein) of RE are addedto the tubes. On the 9th day, the medium is thrown away and replaced byfresh medium; 40 μl of RE are added again. Eight days after the lastaddition of RE, the cultures are treated with a dicolcine salt,commercially available under the trade name "Colcemid" (Gibco), at afinal concentration of 0.25.10⁶ M for 15 hours.

Mitoses stimulated by RE and blocked during metaphases by the action of"Colcemid" are prepared on slides.

After 15 hours contact with "Colcemid" the slides are washed with PBS,removed from the tubes and placed in 6 cm diameter Petri dishes (Falcon1007), so that the development of cells during the treatment can befollowed with a reversed phase contrast microscope.

The slides are covered with 2 ml hypotonic medium of the followingcomposition:

KCl 0.075M: 25 vol.

distilled H₂ O: 25 vol.

Injectable hyaluronidase (Choay): 3 vol.

This solution is preheated for 30 minutes at 37° C. The dishescontaining the slides are placed in the oven at 37° C. for 30 minutes.The hyaluronidase enables the aqueous medium to penetrate the cellsundergoing mitosis and causes them to swell. The dishes are then removedfrom the oven and the following fixative is added:

absolute methyl alcohol: 3 volumes

glacial acetic acid: 1 volume.

This solution is added drop by drop, very slowly to obtain a dilution of1:1 in the hypotonic medium in about one hour.

The dishes are then returned to the oven at 37° C. for 10 minutes. Thehypotonic medium-fixative mixture is then removed from the dishes andreplaced by pure fixative for 20 minutes.

This very slow fixing enables the alcoholic solution to graduallyreplace the water in the swollen cells.

The slides are removed from the dishes, placed on a metal carryingmeans, dried in the oven at 37° C. for one hour and stained with Giemsafor ten minutes.

Giemsa (RAL): 4 volumes

Sorensen phosphate buffer, pH 6.7: 4 volumes

distilled H₂ O: 92 volumes

To obtain R bands on the preparations, the heat denaturation techniquewas used (DUTRILLAUX et al. 1972, CHARPENTIER et al. 1972). For this,the unstained slides are placed in a tube containing an Earle saltsolution, pH 6.5 at 87° C. The tubes are maintained in a water bath atthis temperature for a time varying in a manner inversely proportionalto the age of the preparations. For 15 days old preparations this timeis approximately thirty minutes.

The slides are then removed from the tubes, rinsed with distilled waterand stained with the Giemsa stain previously used.

After 10 minutes staining, the slides are rinsed in demineralized water,blotted between two filter papers and left to dry in the air for twohours.

The slides are fixed with DPX (GURR) onto histology slides.

PREPARATION OF CHROMATID EXCHANGES

Preparation and the starting of cultures are identical to thosedescribed for chromosomic preparations. On the 8th day, the culturemedium is thrown away and replaced by fresh medium containingbromodeoxyuridin (BUdR Sigma) at a final concentration of 0.5 μg per ml.40 μl (400 μg) of protein) of RE are added to each tube and these arereplaced in the oven at 37° C. On the 9th day the medium is thrown awayand replaced by medium also containing BUdR at the same concentration.The same amount of RE is added. Eighteen hours later, the cultures aretreated with Colcemid (Gibco): 0.25 10⁶ M as before, for 15 hours.

The preparation of metaphases is identical to that described above.

The BUdR is incorporated during two consecutive cycles in the place ofthymidin in the ADN of a majority of cells, stimulated by the RE. Themetaphasic chromosomes, after the second replication cycle, have onechromatid bisubstituted by BUdR and one monosubstituted chromatid. Thelatter fixes the Hoechst 33258 fluorochrome, which is then replaced byGiemsa. The technique used is derived from the DE WEERDKASTELEIN et al.(1977) technique, itself adapted from the method described by PERRY etal. (1974). The slides are plunged into a Hoechst 33258 (Hoechst)fluorochrome solution at 0.5 μg per ml of PBS for 15 minutes. They arethen placed, cells down, in 6 cm diameter Petri dishes containing afilter paper moistened with PBS under an UV lamp (254 nm) for one night.

The cells are then placed in a water bath at 61° C. for one hour, intubes containing a buffer:

KCl: 0.3M

Sodium citrate: 0.03M

The slides are then rinsed in distilled water, stained with Giemsa,dried and mounted as above.

Chromosome observation and counting and chromatid exchanges wereeffected with an orthoplaned microscope with an immersion objective.

EXAMPLE I Increase in cellular density and the area covered with adulthuman epidermal keratinocytes after treatment with retinian extract (RE)

In this representative experiment, a 2 to 3 cm² specimen of skin wasremoved from the thoracic area of a 30 years old woman. This specimenwas cut into thinned 2 to 4 mm² fragments. These fragments wereincubated one night in 0.25% trypsin in a phosphate buffer salt (PBS) at+4° C. Once they had dissociated, the epidermis and the derm were gentlyagitated in PBS to obtain the cells. After filtration through two-plygauze, the cells were sedimented at 800 r.p.m. for 10 minutes. Theculture medium (T.C.M.) consisted of the modification of Dulbecco ofEagle MEM in a Hepes buffer with 20% foetal calf serum added. Tissueculture plates (COSTAR-3524) with 24 holes (2 cm² per hole), were seededwith various cell concentrations.

Bovine retinas were used to prepare RE as follows:

Ox eyes are removed at the slaughter house a few moments after theanimals are dead. They are transported on ice and dissection is carriedout within the following 3 hours. After the eyeball has been cleaned bya jet of alcohol at 70%, the cornea is cut and the crystalline lens, theiris and the vitreous humour are placed in separate containers on ice.The retina is then removed with forceps and washed in a neutral isotonicbuffer solution. The retinas are then collected together.

All the consecutive steps of extracting the RE are carried out undercold.

50 retinas are put into 50 ml of PBS buffer and then ground in a Potter.A first centrifugation is effected at 11,000 r.p.m. for 30 minutes. Thesupernatent is then directly filtered through a succession of"Millipore" filters with decreasing pore diameter: 3μ, 1.2μ, 0.45μ,0.22μ, the last providing sterilization. The RE is conserved frozen.

RE can also be obtained, with similar yields, by performing:

(1°) An additional centrifugation at 100,000 g before the filtrations,

(2°) a dialysis against the PBS buffer for one night, followed by thesterilization filtration.

The protein solution was adjusted to 5 mg per ml with respect to PBS.The protein content was measured by the technique described by BRADFORDM. M., Analytical Biochem. 72, 248-254 (1976) using bovine albumin serum(fraction V) as a standard, 20 μl per hole (0.5 ml of TCM) were addedeach day, as follows: At 24 h the RE was added, the TCM was removed onthe 2nd day and replaced by TCM+RE. On day 3, the RE was added withoutchanging the RE. On day 4, the TCM was removed and replaced by freshTCM+RE. On days 5 and 7, the cultures were treated as on day 3, on days6 and 8, as on day 4. The controls were treated in a similar way butwithout the R.E.

From left to right the cells were seeded at rates of:

(a) 5×10⁴

(b) 10⁵

(c) 2×10⁵

(d) 4×10⁵

(e) 6×10⁵

(f) 8×10⁵

cells per hole. The effect of RE on the surface covered by epidermalcells is clearly visible in rows (a), (b) and (c). The increase incellular density can be seen also in rows (c), (d), (e) and (f).

EXAMPLE 2 Increase in mitotic activity as a result of treatment with RE

The mitotic figures were counted in the cultures. For this, 5microscopic fields were examined under a magnification of ×250, in eachhole. The number of mitotic figures per microscopic field (ordinates)was marked as a function of the concentration of seeded cells per hole(abscissa). Each point on the curve represented the mean value of 12counts (3 microscopic fields×4 holes). In the control cultures, about 3mitotic figures could be detected in one microscopic field for a seedingconcentration of 4, 6.4 and 8×10⁵.

In the cultures treated with RE the number of mitotic figures is atleast doubled.

EXAMPLE 3 Accumulation of mitoses for chromosomic charts.

For the chromosomic charts, specimens of skin were taken from 6 adultsubjects. The cell suspensions were prepared as was described in exampleI and adjusted to 2×10⁵ cells per ml in TCM. 2 ml of these suspensionswere seeded on 11×22 m (about 2 cm²) glass slides in Leighton tubes. The8th and 9th days, 100 μg (protein equivalent) per ml of RE were added toeach tube. 12 hours after the 2nd addition of RE, the cells were treatedwith Colcemid (0.25×10⁶ M) for 15 hours to accumulate the metaphases.The slides were rinsed with PBS and transferred into dishes half filledwith hypotonic solution (KCl 0.075M:H₂ O:1:1) to which was added 6%hyaluronidase (Choay). After 30 minutes at +37° C., the fixative(methanol, acetic acid 3:1) was added to the hypotonic solution veryslowly, drop by drop, to obtain a 1:1 dilution in about 1 hour. Thismixture was replaced after 10 minutes at +37° C. by pure fixative for 30minutes at ambient temperature.

EXAMPLE 4 Exchanges of sister chromatids on adult human keratinocytes

Adult human keratinocyte cultures (4 trials) were started at aconcentration of 4×10⁵ cells per glass slide in a Leighton tube, as inexample 3. The cells were cultured for 8 days in normal TCM, without RE,as in example I. On the 8th day, the TCM was removed and replaced by TCMcontaining BUdR (final concentration 0.2 μg per ml). 100 μg (proteinequivalent) of RE was added. After 24 hours at +37° C., the medium wasremoved and replaced by fresh TCM containing the same amount of BUdR andRE respectively.

The cells were incubated under these conditions for 36 hours. Aftertreatment with Colcemid, the hypotonic solution and the fixative, as isshown in FIG. 3, the cultures were stained with Hoechst 33,358 (0.5 μgper ml in PBS) for 15 minutes. Then, after exposure to U.V. light at 254nm for one night, the slides were treated with SSC×2 (0.3M KCl, 0.03Msodium citrate) at +61° C. for one hour (DE WEERD-KASTELEIN E. A.,KEIZER W., RAINALDI G. and BOOTSMA D. Mutat. Res. 45 253-261 (1977).

The cells were rinsed with water and stained with 4% Giemsa in phosphatebuffer pH 6.7 for 10 minutes.

EXAMPLE 5 Comparison of the activity of RE and that of another growthfactor (FGF)

The following example is given to demonstrate the difference in theactivity of RE and of the factor FGF on myoblast growth (see D.Gospodarowicz's article mentioned hereinabove).

Myoblasts prepared by the dissociation of calf member muscles are seededin Petri dishes. The culture medium only contains 0.5% serum. For 2days, the cells receive 50 μl of RE or 10 ng of F.G.F. The number ofcells is determined directly on half of the dishes; on the other half,incorporation of radioactive thymidine is measured after 4 hoursincubation. The results show that the control contains 40,000 cells and8100 Cpm, whereas the sample treated with RE contains 120,000 cells and41,000 Cpm and the sample treated with FGF contains 40,000 cells and25,000 Cpm.

Therefore, there is marked stimulation due to RE. In this case, the FGFdoes not modify the number of cells, as compared with the control.

EXAMPLE 6

This example relates to the action of the RE factor on the growth ofcultured guinea pig epidermal cells.

The cells were obtained from the ear of an adult Hartley guinea pig(class IV). Three types of cells were isolated:

(a) basal epidermal cells,

(b) suprabasal epidermal cells,

(c) conjunctive dermic tissue cells,

The epidermal cells were seeded at three different concentrations:500,000/cm² -250,000/cm² and 100,000/cm².

The fibroblastic cells were seeded at 50,000/cm² and at 400,000/cm².

All the cultures were made in an Eagle BME+10% foetal calf serum medium.The cells were cultured in triplicate in plastic Petri dishes.

The RE was added to the culture medium at a rate of 100 μg proteinequivalent per ml. The cells were seeded in a culture medium containingRE. The media were changed on the 1st, 3rd, 5th and 8th days andreplaced by fresh media also containing the same rate of RE. The cellswere therefore maintained in the presence of RE during the entireexperiment.

Tritiated thymidine (³ H-methyl-thymidine Amersham a. s.40 Ci/mM) wasadded to the final concentration of 5 μCi/ml.

After incubation for one hour, the medium was removed, the cells werewashed once in PBS, then in 0.3M potash directly in the Petri dish forone hour at 37° C. The viscous solution obtained was precipitated withPCA at 70%. The precipitate was washed three times with 0.4M PCA andhydrolyzed in 0.5M PCA for 15 minutes at 95° C.

A supernatent aliquot was counted for radioactivity and another aliquotwas used to measure its DNA content according to the Burton technique.

In order to assess desquamation on days 1,3,5 and 8, the cell culturemedia were recovered and filtered through 0.45μ weighed Milliporefilter. 48 hours later, the air-dried filters were weighed again. Thedry weight obtained corresponded to the protein content in mg.

RESULTS

The trials reported above show that the RE has a significant effect onDNA synthesis by basal cells on the 8th day at 2×10⁶ cells, i.e. at therate of 250,000 cells/cm². The effect is less constant on the same cellsat 500,000 per cm².

The induction effect is especially clear on the 8th day on mature(suprabasal) cells seeded at a rate of 250,000/cm².

Finally, no effect was observed at 8 days on derm cells (mainlyfibroblasts).

Furthermore, desquamation is not modified.

On the whole RE was shown to be an activator in epidermal cells(basal+suprabasal) seeded at 250,000 per cm² after the cells have beenexposed to the product for 8 days. Under the same conditions, the actionwas non-existant for dermic cells.

It is seen, therefore, that RE has a stimulating effect on the in vitrogrowth of adult epidermal cells both in man and in the guinea pig.

EXAMPLE 7

Additional experiments were conducted to demonstrate the absence of sideeffects of RE.

Study on the mutagen activity of RE on various strains of Salmonellatyphimurium and at concentrations of 22.5 to 720 μg/culture, showed nomutagen effect according to the Ames method.

At a concentration of 100 μg/ml, RE caused no local irritation of theskin.

The irritation rating on the ocular conjunctiva, at a concentration of 5mg/ml, also showed no irritant effect for rabbit eye.

We claim:
 1. A process for stimulating the growth of epiderm cells, which comprises contacting the epiderm cells with an aqueous saline extract of ocular tissue (RE).
 2. The process according to claim 1, wherein the active agent is extracted from eye tissues inclusive of the choroid, the iris and the vitreous humour.
 3. The process according to claim 2, wherein the RE is obtained by the treatment of bovine ocular tissues with a buffered aqueous saline solution with a pH of about 7.2.
 4. The process according to claim 3, wherein there is used 50 to 400 μg, in total protein, of extract per ml of epidermal cell culture.
 5. The process according to claim 4, wherein the culture is a culture of human epiderm cells.
 6. The process according to claim 5 wherein said human epiderm cell is suitable for the detection of natural or induced chromosome anomalies.
 7. The process according to claim 6 wherein said human epiderm cell is suitable for the assessment of sister chromatid exchanges (SCE) in in vitro cultures of adult human keratinocytes.
 8. The process according to claim 7, which further comprises first effecting a preliminary normal culturing of epidermal cells in the absence of the growth factor RE, and then introducing therein after a period of about 5 to 10 days, an effective amount of RE and, then introducing about one or two days later, another effective amount of RE and obtaining a second replication of the cells.
 9. The process according to claim 7, wherein the culturing of epidermal cells is effected by introducing, at the beginning, an effective amount of RE and then arresting the culturing to permit cell synchronization.
 10. The process according to claim 5 resulting in grafts.
 11. The process according to claim 10 which further comprises integrating said process into a grafting technique comprising removing small fragments of skin for a subsequent autograft, splitting the graft by enzymatic means 1 to 2 weeks before the culture, causing the cultured epidermal cells to proliferate on a substrate comprising thin flaps of human skin or pig skin, the RE being added in an effective amount for quick cultivation of epidermal cells, and effecting the graft in a conventional manner.
 12. The process of anyone of claims 1 to 5 comprising the long-term culturing of epidermal cells.
 13. The process according to claim 12, wherein the cells are cultured directly on a substrate in the presence of an effective amount of RE and without the addition of other cells.
 14. The process of claim 10 wherein said graft is suitable for treatment of burns. 